Staphylocuccus aureus is a major human pathogen that causes a range of diseases. The incidence of staphylococcal infections continues to rise. Current methods of treatment rely almost exclusively on antibiotic therapy. Biofilm formation is a major contributor of S. aureus virulence because it protects the bacteria from antibiotics or host immune system. Specifically, biofilms provide intrinsic resistance to antibiotics and protection from host defenses, which leads to treatment failures. Biofilms consist of multiple layers of bacterial cells contained within an extracellular matrix that keeps the biofilm intact. Understanding the nature of biofilm matrix could lead to new methods to prevent biofilm formation or eradicate bacteria within a biofilm, thus providing a way to overcome the therapeutic recalcitrance of biofilm-associated infections. Factors previously shown to contribute to biofilm formation in S. aureus include surface-associated proteins, exopolysaccarides, and, extracellular DNA, but to date the contribution of these components to staphylococcal biofilm-associated infections has not been defined. More importantly, we do not fully understand the composition of biofilm matrix, especially the protein components, in biofilms formed during an infection. The goal of this application is to identify proteins in biofilms from an infection site sing a suitable animal model. To this end, we have recently developed a rat model of implant-associated orthopedics infection allowing us to isolate sufficient bacteria from an infection site o identify bacterial components directly using an advanced proteomic method. Thus, in this application, we propose to comprehensively identify proteins in biofilm matrix produced under in vivo conditions (Aim 1) and to validate the presence of a set of prioritized individual components in the infection site (Aim 2).